共查询到20条相似文献,搜索用时 15 毫秒
1.
Wanli Liu Pengjun Shi Qiang Chen Peilong Yang Guozeng Wang Yaru Wang Huiying Luo Bin Yao 《Applied biochemistry and biotechnology》2010,162(1):1-12
A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques.
The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The
deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml−1. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at
acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The
specific activity, K
m, and V
max for oat spelt xylan substrate was 7,988 U mg−1, 22.2 mg ml−1, and 15,105.7 μmol min−1 mg−1, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications. 相似文献
2.
Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of d-glucose at carbon 2 in the presence of molecular O2 producing d-glucosone (2-keto-glucose and d-arabino-2-hexosulose) and H2O2. It was used to convert d-glucose into d-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several
parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature,
nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography
on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of d-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion.
On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H2O2 acted as inhibitor for this reaction. The rate of bioconversion of d-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO2 at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55°C) and
pH (5.0) of d-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation
energy (E
a) was 32.08 kJ mol−1 and kinetic parameters (V
max, K
m, K
cat and K
cat/K
m) for this bioconversion were 8.8 U mg−1 protein, 2.95 mM, 30.81 s−1 and 10,444.06 s−1 M−1, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of d-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect
significantly the enzyme activity. 相似文献
3.
Javed MR Rashid MH Nadeem H Riaz M Perveen R 《Applied biochemistry and biotechnology》2009,157(3):483-497
Monomeric extracellular endoglucanase (25 kDa) of transgenic koji (Aspergillus oryzae cmc-1) produced under submerged growth condition (7.5 U mg−1 protein) was purified to homogeneity level by ammonium sulfate precipitation and various column chromatography on fast protein
liquid chromatography system. Activation energy for carboxymethylcellulose (CMC) hydrolysis was 3.32 kJ mol−1 at optimum temperature (55 °C), and its temperature quotient (Q
10) was 1.0. The enzyme was stable over a pH range of 4.1–5.3 and gave maximum activity at pH 4.4. V
max for CMC hydrolysis was 854 U mg−1 protein and K
m was 20 mg CMC ml−1. The turnover (k
cat) was 356 s−1. The pK
a1 and pK
a2 of ionisable groups of active site controlling V
max were 3.9 and 6.25, respectively. Thermodynamic parameters for CMC hydrolysis were as follows: ΔH* = 0.59 kJ mol−1, ΔG* = 64.57 kJ mol−1 and ΔS* = −195.05 J mol−1 K−1, respectively. Activation energy for irreversible inactivation ‘E
a(d)’ of the endoglucanase was 378 kJ mol−1, whereas enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) of activation at 44 °C were 375.36 kJ mol−1, 111.36 kJ mol−1 and 833.06 J mol−1 K−1, respectively. 相似文献
4.
Wei Zhao Aisheng Xiong Xiaoyan Fu Feng Gao Yongsheng Tian Rihe Peng 《Applied biochemistry and biotechnology》2010,162(8):2157-2165
To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni2+–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC)
had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being
incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg−1, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg−1 min−1, respectively. The enzyme activity was significantly improved by 1 mM of K+, Ca2+, and Mg2+. These characteristics contribute to its potential application in feed industry. 相似文献
5.
A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein
is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg−1 at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a
wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K
m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced
by Na+, NH4+, Mg2+, Fe2+, Fe3+, Co2+, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents. 相似文献
6.
A 66-kDa thermostable family 1 Glycosyl Hydrolase (GH1) enzyme with β-glucosidase and β-galactosidase activities was purified
to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family. N-terminal and partial internal amino acid sequences showed significant resemblance to plant GH1 enzymes. Kinetic
studies showed that enzyme hydrolyzed p-nitrophenyl β-d-glucopyranoside (pNP-Glc) with higher efficiency (K
cat/K
m = 2.27 × 104 M−1 s−1) as compared to p-nitrophenyl β-d-galactopyranoside (pNP-Gal; K
cat/K
m = 1.15 × 104 M−1 s−1). The optimum pH for β-galactosidase activity was 4.8 and 4.4 in citrate phosphate and acetate buffers respectively, while
for β-glucosidase it was 4.6 in both buffers. The activation energy was found to be 10.6 kcal/mol in the temperature range
30–65 °C. The enzyme showed maximum activity at 65 °C with half life of ~40 min and first-order rate constant of 0.0172 min−1. Far-UV CD spectra of enzyme exhibited α, β pattern at room temperature at pH 8.0. This thermostable enzyme with dual specificity
and higher catalytic efficiency can be utilized for different commercial applications. 相似文献
7.
A laccase has been purified from the liquid culture growth medium containing bagasse particles of Fomes durissimus. The method involved concentration of the culture filtrate by ultrafiltration and anion exchange chromatography on diethyl
aminoethyl cellulose. The sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide
gel electrophoresis both gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the
purified laccase determined from SDS-PAGE analysis was 75 kDa. Using 2,6-dimethoxyphenol as the substrate, the determined
K
m and k
cat values of the laccase are 182 μM and 0.35 s−1, respectively, giving a k
cat/K
m value of 1.92 × 103 M−1 s−1. The pH and temperature optimum were 4.0 and 35 °C, respectively. The purified laccase has yellow colour and does not show
absorption band around 610 nm found in blue laccases. Moreover, it transformed methylbenzene to benzaldehyde in the absence
of mediator molecules, property exhibited by yellow laccases. 相似文献
8.
Yao Guo Fengxia Lu Haizhen Zhao Yanchong Tang Zhaoxin Lu 《Applied biochemistry and biotechnology》2010,162(2):498-509
A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of
605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the
AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around
94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions
after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum
temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below
40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting
revealed that rGOD exhibited a K
m value of 16.95 mM and a K
cat value of 484.26 s−1. 相似文献
9.
Hossein Amani Mohammad Reza Mehrnia Mohammad Hossein Sarrafzadeh Manouchehr Haghighi Mohammad Reza Soudi 《Applied biochemistry and biotechnology》2010,162(2):510-523
There is a lack of fundamental knowledge about the scale up of biosurfactant production. In order to develop suitable technology
of commercialization, carrying out tests in shake flasks and bioreactors was essential. A reactor with integrated foam collector
was designed for biosurfactant production using Bacillus subtilis isolated from agricultural soil. The yield of biosurfactant on biomass (Y
p/x), biosurfactant on sucrose (Y
p/s), and the volumetric production rate (Y) for shake flask were obtained about 0.45 g g−1, 0.18 g g−1, and 0.03 g l−1 h−1, respectively. The best condition for bioreactor was 300 rpm and 1.5 vvm, giving Y
x/s, Y
p/x, Y
p/s, and Y of 0.42 g g−1, 0.595 g g−1, 0.25 g g−1, and 0.057 g l−1 h−1, respectively. The biosurfactant maximum production, 2.5 g l−1, was reached in 44 h of growth, which was 28% better than the shake flask. The obtained volumetric oxygen transfer coefficient
(K
L
a) values at optimum conditions in the shake flask and the bioreactor were found to be around 0.01 and 0.0117 s−1, respectively. Comparison of K
L
a values at optimum conditions shows that biosurfactant production scaling up from shake flask to bioreactor can be done with
K
L
a as scale up criterion very accurately. Nearly 8% of original oil in place was recovered using this biosurfactant after water
flooding in the sand pack. 相似文献
10.
S. Subramaniyan 《Applied biochemistry and biotechnology》2012,166(7):1831-1842
Low molecular weight endo-xylanase from Bacillus pumilus SSP-34 was purified to homogeneity using ion exchange and size exclusion chromatographies. Xylanases were isolated by novel
purification protocol which includes the use of anion exchange matrix such as DEAE Sepharose CL 6B with less affinity towards
enzyme protein. The purified B. pumilus SSP-34 have a molecular weight of 20 kDa, with optimum pH and temperature at 6.0 and 50 °C, respectively. The enzyme was
stable at 50 °C for 30 min. It showed remarkable stability at pH values ranging from 4.5 to 9 when the reaction was carried
out at 50 °C. K
m and V
max values, determined with oats spelts xylan were 6.5 mg ml−1 and 1,233 μmol min−1 mg−1 protein, respectively, and the specific activity was 1,723 U mg−1 相似文献
11.
Meijuan Xu Zhiming Rao Hong Xu Chunyan Lan Wenfang Dou Xiaomei Zhang Hongyu Xu Jian Jin Zhenghong Xu 《Applied biochemistry and biotechnology》2011,163(6):707-719
Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial l-arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g−1, and the oxygen uptake rates reached 0.25 mg A562−1 h−1 which enhanced 38.8% compared to the wild-type strain. Thus, the final l-arginine concentration of the batch fermentation reached a high level of 35.9 g L−1, and the biomass was largely increased to 6.45 g L−1, which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the
first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for l-arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products. 相似文献
12.
Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified
enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima
at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from
40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0
for 24 h. The apparent K
m and V
max of Chi72 for colloidal chitin were 0.23 mg ml−1 and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the
recycling of chitin waste. 相似文献
13.
Junpei Zhou Huoqing Huang Kun Meng Pengjun Shi Yaru Wang Huiying Luo Peilong Yang Yingguo Bai Bin Yao 《Applied biochemistry and biotechnology》2010,160(5):1277-1292
A bacterial strain, Streptomyces sp. TN119, was isolated from the gut of Batocera horsfieldi larvae and showed xylanolytic activity. A degenerate primer set was designed based on the base usage of G and C in Actinobacteria xylanase-coding sequences belonging to the glycosyl hydrolases family 10 (GH 10), and used to clone the partial xylanase
gene from Streptomyces sp. TN119. A modified thermal asymmetric interlaced (TAIL)-PCR specific for high-GC genes, named GC TAIL-PCR, was developed
to obtain the full-length xylanase gene (xynA119; 1089 bp). Rich in GC content (67.8%), xynA119 encodes a new GH 10 xylanase (XynA119), which shares highest identity (48.8%) with an endo-1,4-β-xylanase from Cellulosimicrobium sp. HY-12. Recombinant XynA119 was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 6.5 and 60 °C, was stable
at pH 4.0 to 10.0 and 50 °C, was resistant to most chemicals (except for Cu2+, Mn2+, Ag+, Hg2+ and SDS) and trypsin, and produced simple products. The specific activity, K
m, V
max, and k
cat using oat-spelt xylan as substrate were 57.9 U mg−1, 1.0 mg ml−1, 74.8 μmol min−1 mg−1, and 49.2 s–1, respectively. 相似文献
14.
An exoinulinase has been isolated, purified and characterised from a commercially available broth of Aspergillus ficuum. The enzyme was purified 4.2-fold in a 21% yield with a specific activity of 12,300 U mg−1(protein) after dialysis, ammonium sulphate fractionation and Sephacryl S-200 size exclusion and ion exchange chromatography.
The molecular weight of this enzyme was estimated to be 63 kDa by SDS-PAGE. It exhibited a pH and temperature optima of 5.4
and 50 °C respectively and under such conditions the enzyme remained stable with 96% and 63.8% residual activity after incubation
for 12 h and 72 h respectively. The respective K
m and V
max values were 4.75 mM and 833.3 μmol min−1 ml−1, respectively. Response surface methodological statistical analysis was evaluated for the maximal production of fructose
from the hydrolysis of pure commercial chicory inulin. Incubation of the dialyzed crude exoinulinase (100 U/ml, 48 h, 50 °C,
150% inulin, pH 5.0) produced the highest amount of fructose (106.4 mg/ml) under static batch conditions. The purified exoinulinase
was evaluated for fructose production and the highest amount (98 mg/ml) was produced after 12 h incubation at 50 °C, 150%
inulin pH 5.0. The use of a crude exoinulinase preparation is economically desirable and the industrial production of fructose
from inulin hydrolysis is biotechnologically feasible. 相似文献
15.
To investigate the production of cellulases and xylanases from Penicillium echinulatum 9A02S1, solid-state fermentation (SSF) was performed by using different ratios of sugar cane bagasse (SCB) and wheat bran
(WB). The greatest filter paper activity obtained was 45.82 ± 1.88 U gdm−1 in a culture containing 6SCB/4WB on the third day. The greatest β-glucosidase activities were 40.13 ± 5.10 U gdm−1 obtained on the third day for the 0SCB/10WB culture and 29.17 ± 1.06 U gdm−1 for the 2SCB/8WB culture. For endoglucanase, the greatest activities were 290.47 ± 43.57 and 276.84 ± 15.47 U gdm−1, for the culture 6SCB/4WB on the fourth and fifth days of cultivation, respectively. The greatest xylanase activities were
found on the third day for the cultures 6SCB/4WB (36.38 ± 5.38 U gdm−1) and 4SCB/6WB (37.87 ± 2.26 U gdm−1). In conclusion, the results presented in this article showed that it was possible to obtain large amounts of cellulases
and xylanases enzymes using low-cost substrates, such as SCB and WB. 相似文献
16.
Mervat Morsy A. El-Gendy 《Applied biochemistry and biotechnology》2010,162(3):780-794
Among all endophytic keratinolytic fungal isolates recovered from marine soft coral Dendronephthya hemprichii, Penicillium spp. Morsy1 was selected as the hyperactive keratinolytic strain under solid substrate fermentation of different agriculture
and poultry wastes. The optimization of extraction process, physicochemical parameters affecting the keratinase production
in solid-state fermentation, and the purified keratinase parameters were studied. Maximum keratinase activity (1,600 U g−1, initial dry substrate) was recovered from moldy bran with 0.1% Tween 80. The optimized production conditions were rice straw
as carbon source, pH of medium 6, growth temperature 26 °C, initial moisture content of 80% (v/w), inoculum size of 105 spores ml−1, and an average particle size of the substrate 0.6 mm (3,560 U g−1, initial dry substrate after 5 days of fermentation). Two types of keratinase (Ahm1 and Ahm2) were purified from the culture
supernatant through ammonium sulfate precipitation, DEAE-Sepharose, and gel filtration chromatography. Enzyme molecular weights
were 19 kDa (Ahm1) and 40 kDa (Ahm2). The kinetic parameters of purified keratinases were optimized for the hydrolysis of
azokeratin by Ahm1 (pH 7.0–8.0, stable in pH range of 6.0 to 8.0 at 50 °C) and Ahm2 enzymes (pH 10.0–11.0, stable in pH range
of 6.0 to 11.0 at 60–65 °C). Whereas inhibitors of serine (phenylmethylsulfonyl fluoride) and cysteine (iodoacetamide) proteases
had minor effects on both Ahm1 and Ahm2 activity, both keratinases were strongly inhibited by chelating agents EDTA and EGTA.
These findings suggest that serine and cysteine residues are not involved in the catalytic mechanisms, and they are metalloproteases. 相似文献
17.
18.
Divanery Rodriguez-Gomez Linda Lehmann Nadja Schultz-Jensen Anne Belinda Bjerre Timothy John Hobley 《Applied biochemistry and biotechnology》2012,166(8):2051-2063
Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw
as carbon source which was sterilized by autoclavation. To account for any effects of autoclavation, a comparison was made
with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw
(which contained antibiotics) was best suited for the production of xylanases (110 IU ml−1) and cellulases (0.5 filter paper units (FPU) ml−1). Addition of Avicel boosted enzyme titers with the highest cellulase titers (1.5 FPU ml−1) found with addition of 50 % w/w Avicel and with the highest xylanase production (350 IU ml−1) reached in the presence of 10 % w/w Avicel. Comparison with enzyme titers from other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising
and suitable substrate for cellulase and hemicellulase production. 相似文献
19.
Lawuyi B Chen H Afkhami F Kulamarva A Prakash S 《Applied biochemistry and biotechnology》2007,142(1):71-80
This article demonstrates the potential of encapsulated, engineered Lactococcus lactis as a vehicle for the oral delivery of therapeutic proteins. Using alginate-poly-l-lysine-alginate membrane-encapsulated L. lactis engineered to secrete the reporter protein Staphylococcal aureus nuclease, we show comparable viability and protein secretion between free and immobilized cells. After 12 h, microcapsules
with a cell density of 4.8 × 105 colony forming unit (CFU) ml−1 grew to 2.2 × 108 CFU ml−1 and released 0.24 arbitrary unit (AU) ml−1 of nuclease, producing similar results as free cells, which grew from 3.4 × 105 to 1.9 × 108 CFU ml−1 and secreted 0.21 AU ml−1 of nuclease. Moreover, encapsulated cells at a density of 4.4 × 107 CFU ml−1 grew to 2.2 × 1010 CFU ml−1 in 12 h and secreted 15.3 AU ml−1 of nuclease although 3.1 × 107 CFU ml−1 of free cells reached only 2.3 × 109 CFU ml−1 and released 5.6 AU ml−1 of nuclease. We also show the sustained stability of the microcapsules during storage at 4°C over 8 weeks. 相似文献
20.
A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding
rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l−1) with 100 ml sucrose solution (190 g l−1) being evenly fed (9–10 ml h−1) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions,
the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively.
When the sucrose concentration was controlled at 5–10 g l−1 in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l−1, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved
to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be
scaled up, since various parameters involved in industrial production were taken into account. 相似文献